Optimization of Pulse Electric Field Treatment and Vacuum Impregnation for Reducing Freezing Injury of Thawed Rucola Leaves

Abstract: Freezing injuries usually take place in thawed plant-based food products. To reduce freezing injuries, Pulse Electric Field (PEF) treatment and Vacuum Impregnation (VI) of cryoprotectant were used before the freezing process. The aim of PEF is to provoke electroporation to the membranes of the plant cells, thereby allowing the cryoprotective solution impregnated by VI to penetrate the cells. PEF treatment was optimized to use the minimum possible pulse width in order to obtain a homogeneous electroporation of rucola leaves. In order to achieve this minimum pulse width, several combinations of electric field strengths, pulse numbers, and number of trains were tested. The verification for the electroporation was done first by observing the treated sample with a microscope after it had been stained with Propidium Iodide and then by monitoring the tissue leakage by measuring its conductivity over time. VI treatment was done straight after the PEF treatment and then the sample was stored in a refrigerator to rest before freezing and thawing. The initial experiment attempted to optimize the PEF parameters, while the second experiment was carried out to determine the optimal concentration of cryoprotectant for VI when combined with an optimized PEF treatment. The leaves were then rested, frozen and thawed before being observed. The initial experiment was done using commercial rucola leaves from Italy. Due to the seasonal availability of commercial rucola leaves, the second experiment was done using rucola leaves planted in the greenhouse at the Biology Department of Lund University. The results show that the samples treated with PEF and VI with the highest concentration of the cryoprotectant (SC4) had the highest survival after freezing and thawing. This treatment resulted in 88.41 ± 5.23 % of survival 5 min after leaves were taken out from a freezer and decreased to 57.97 ± 5.80 % after 1 hour. The microscopic observations showed that viable cells of frozen and thawed leaves were mostly located near the main veins.